12q21 Interstitial Deletions: Seven New Syndromic Cases Detected by Array-CGH and Review of the Literature

Interstitial deletions of the long arm of chromosome 12 are rare, with a dozen patients carrying a deletion in 12q21 being reported. Recently a critical region (CR) has been delimited and could be responsible for the more commonly described clinical features, such as developmental delay/intellectual disability, congenital genitourinary and brain malformations. Other, less frequent, clinical signs do not seem to be correlated to the proposed CR. We present seven new patients harboring non-recurrent deletions ranging from 1 to 18.5 Mb differentially scattered across 12q21. Alongside more common clinical signs, some patients have rarer features such as heart defects, hearing loss, hypotonia and dysmorphisms. The correlation of haploinsufficiency of genes outside the CR to specific signs contributes to our knowledge of the effect of the deletion of this gene-poor region of chromosome 12q. This work underlines the still important role of copy number variations in the diagnostic setting of syndromic patients and the positive reflection on management and family genetic counseling.


Introduction
Interstitial deletions in 12q21 have been described in a dozen patients who were all carriers of non-recurrent copy number variants (CNVs) [1][2][3][4][5][6][7][8][9][10]. Despite common features, such as developmental delay and/or intellectual disability (DD/ID), congenital genitourinary, brain malformations and ectodermal anomalies, the clinical presentation is characterized by wide clinical expressivity with severity and signs depending on the size and location of the deletions and on the involved genes. In most cases the deletions are large, making it difficult to assign the causative genes to individual phenotypic signs.
In this study we describe seven new patients with deletions detected by array-based comparative genomic hybridization (array-CGH), collected through a cooperation organized by the Cytogenetics and Cytogenomics Working Group of the Italian Society of Human Genetics (SIGU). The involved regions spanning from 1 to 18.53 Mb are differentially positioned in the 12q21.1-q21.33 region. Three deletions overlap the 1.6 Mb CR in 12q21.2, confirming its potential causality for the aforementioned clinical features. The other four deletions, relatively smaller (1 Mb to 6.75 Mb), map distally, hence providing the tool to investigate the contribution of genes outside the CR on the clinical phenotype. Indeed, the carrier patients manifest rare but recurrent clinical signs such as heart defects, hearing loss, hypotonia and facial dysmorphisms including down-slanting palpebral fissures, wide mouth and prominent forehead.
The genotype-phenotype correlations in our patients, compared with cases in the literature and in the DECIPHER database, bring to light relationships between the deletion of specific 12q21 genes and distinct phenotypic traits.

Array-CGH
Our study includes seven patients, five males and two females, with age at diagnosis ranging from 4 to 16 years. Array-CGH was performed on all seven patients and the results are provided in Figure 1 (red bars) and Figure 2, while the clinical features of each different 12q microdeleted patient are detailed in Table 1. Patients' 2 and 7 deletions have been confirmed by BAC-FISH experiments and Figure 2 shows the images for Patient 2. Patients 2 and 3 have been extrapolated from a cohort of 5110 Italian patients, collected by the SIGU Cytogenetics and Cytogenomics Working Group and analyzed by array-CGH as reported by Catusi et al. (2020) [16].
The inheritance pattern could be determined in four of the seven patients: the microdeletion arose de novo in Patients 5, 6 and 7 and was transmitted to Patient 2 by her mother, also with mild intellectual disability and minor dysmorphisms.
The extent of microdeletions ranges from the smallest 1 Mb deletion of Patient 7 in 12q21.32q21.33, only containing five coding genes, to the largest 18.5 Mb deletion of Patient 1 in 12q21.1q21.33, comprising 46 coding genes ( Figure 1 and Table 1).
None of the deleted regions overlap with established benign losses reported in the Database of Genomic Variants (DGV) [17].     Learning and language impairments.
Mild ID and language impairment.

12q21 Region and Synopsis of Relevant Genes
The 12q21 region spans 21.2 Mb and includes 60 coding genes with a gene density remarkably lower relative to the entire chromosome 12 (2.8 vs. 7.5 coding genes/Mb, respectively). Such a gene density is below even that of chromosome 21, which is considered the poorest gene-containing chromosome (4.5 coding genes/Mb) [13]. To predict the deleterious effect of a deletion, the metrics of haploinsufficiency (HI) and loss-of-function intolerance (pLI) were examined, and 20 of the 60 coding genes in 12q21 stood out as most interesting. Genes with HI score ≤ 10% and/or pLI score ≥ 0.9 are listed in Supplementary Table S1 that also includes disease-causing genes matching the clinical features of our and literature patients.
A number of 12q21 deletions are reported in the DECIPHER database. We have extrapolated the most informative ones according to patients' phenotype and the region involved. The smallest deletion, sized 257 kb, only involves PPP1R12A (Patient 291358), while the largest spans 11 Mb and includes 50 coding genes (Patient 1875) [13]. Figure 1 reports the deletions known from the literature (black bars), the DECIPHER deletions (purple bars), and the seven deletions reported in the present study (red bars). Patients' clinical features are shown in Table 2 for literature patients and in Table 3 for DECIPHER patients.

Discussion
We surveyed seven new patients carrying microdeletions sized from 1 Mb to 18.53 Mb, differentially positioned in the 12q21.1-q21.33 region. All patients have DD/ID, which is confirmed as the most common clinical sign. In three patients (1, 2 and 3) the microdeletions partially overlap the 1.6 Mb CR recently proposed, and support the recurrent clinical signs, namely, DD/ID, growth retardation, brain and genitourinary defects and ectodermal anomalies, associated with 12q21 deletions [5]. The CR is currently the minimal overlapping region among literature patients. Since the reported deletions are mainly wide, spanning from 5.5 to 25.3 Mb, the association of the deletion of specific 12q21 genes to distinct phenotypic traits is still a challenge.
As mentioned above, the proposed CR encompasses four genes: SYT1, PAWR, PPP1R12A and OTOGL, with SYT1 and PPP1R12A as the most convincing candidates [5].
SYT1 encodes for Synaptotagmin 1, a protein highly and exclusively expressed in the brain and involved in trafficking of synaptic vesicles at synapses. A SYT1 HI score of 3.9% is far below the upper threshold (10%) fixed for haploinsufficiency [13,15]. Heterozygous SYT1 mutations are associated with Backer-Gordon syndrome (BAGOS; OMIM #618218) which is characterized by DD, motor impairment and dysmorphisms such as high forehead, smooth philtrum and short nose [18]. The same features recur in 12q21 deletion patients including the SYT1 gene. The only patient with a small deletion affecting the sole SYT1 gene is DECIPHER Patient 331592, who shows only DD/ID (Figure 2 and Table 3).
PPP1R12A, a developmental gene encoding the regulatory subunit 12 of phosphatase, involved in cell migration, adhesion, and morphogenesis, has a HI score of 2.14% and a pLI score of 1, hence being placed at the top of the dosage sensitive genes within the CR. PPP1R12A loss-of-function heterozygous pathogenic variants have been associated with a congenital malformation syndrome affecting the embryogenesis of genitourinary and brain systems (GUBS; OMIM *618820) including sex development disorders. Carriers also show dysmorphisms such as micro-or macrocephaly, upslanting palpebral fissures, long philtrum, micrognathia, low-set ears, hypertelorism, strabismus and small nose [19]. No sex development disorders have been recorded for 12q21 deletions involving PPP1R12A, but brain and genitourinary defects are recurrent. Among our patients, Patient 2 has horseshoe kidney and several genitourinary defects, as already reported in literature patients [2,5,7]. Moreover, the same dysmorphisms are present in all reported patients whose deletions include PPP1R12A, and in DECIPHER Patients 1582 and 291358. This latter patient carries a 257 kb deletion involving PPP1R12A exons from 2 to 25.
The deletions of our Patients 1, 2 and 3 confirm the CR by Niclass et al. [5], including the two major candidate genes. Furthermore, clinical data are consistent with a concurrent effect of SYT1 and PPP1R12A deletions, which are likely to be mainly responsible for the neurodevelopmental issues and for the congenital defects and dysmorphism, respectively. Further single gene deletions and functional studies could help to assess the sensitivity of different tissues to the loss of either gene, and to further investigate their role in clinical signs etiology.
However, Patients 4, 5, 6 and 7 manifest DD/ID, although their deletions do not overlap with the CR region. It could be that other genes included in these deletions could be implicated in DD/ID onset, such as TMTC2, SLC6A15, CEP290, TMTC3 and NTS, which are highly expressed in the brain [12] and predicted as dosage-sensitive by HI or pLI scores (Supplementary Table S1).
Conversely, on the basis of current knowledge, SYT1 and PPP1R12A do not seem related to the ectodermal anomalies observed in 12q21 deleted patients and, moreover, none of the genes identified appear to be specifically expressed in the skin. Considering the deletions of patients showing ectodermal anomalies, we hypothesize that the causative gene(s) for skin features resides within a region of about 4.7 Mb comprised between the start of the deletion of our Patient 3 and the end of the deletion of Patient 3 of Niclass et al. [5] (nucleotides 78634011-83378230; Figure 2). Further studies and patients are needed to corroborate this hypothesis.
Patients 4 and 6 also present interatrial defects (Table 1) consistent with some literature patients [2,4,7,8] and DECIPHER Patient 261816. Overlap of these patients' deletions point to TMTC2 and SLC6A15 as candidate genes, though no expression or mutation data are yet available to associate these genes to cardiac defects.
Our Patients 5 and 6 carrying deletions involving NTS and SLC6A15 manifest DD/ID associated with facial dysmorphisms. Patient 5 has a broad nasal base, low-hanging columella, wide mouth, down-slanting palpebral fissures, while Patient 6 has a wide mouth. Interestingly, Akilapa et al. [9] have described three members of the same family with a varying degree of ID, facial dysmorphisms as wide mouth, broad nasal base with low-hanging columella, tall stature and obesity, segregating with a 2.2 Mb deletion in 12q21.31q21.32. The Authors proposed NTS and SLC6A15 as candidate genes for ID, since both are expressed in the brain and NTS has a predicted HI score of 1.63% (Supplementary  Table S1). It is noteworthy that ALX1, a gene mapping between SLC6A15 and NTS, is associated with autosomal recessive frontonasal dysplasia 3 (FND3, OMIM #613456), a syndrome of variable severity, characterized by a series of facial abnormalities affecting eyes, forehead and nose [20]. The ALX1 gene can be thus proposed as a plausible candidate for the facial dysmorphisms observed in 12q21.31q21.32 deletion carriers, even though evidence of a facial phenotype has not yet been assessed in the case of heterozygous deletions. It will be useful in the future to more precisely describe patients with overlapping deletions in order to better delineate a dysmorphic phenotype resulting from heterozygous loss of function of ALX1 and/or NTS.
For Patient 5, WES was also performed, revealing an additional likely pathogenic loss-of-function homozygous variant in the ACY1 gene localized in 3p21.2: c.1057C>T (p.Arg353Cys), inherited from both parents. ACY1 encodes for the amino acylase 1 enzyme. Its deficiency results in a low hydrolysis of a series of N-acetyl-L-amino. The clinical manifestation of ACY1 deficiency is heterogeneous and includes muscular hypotonia, brain malformations, ID and other neurologic symptoms, while normal psychomotor development has also been noted in some individuals [21]. ACY1 deficiency could explain DD/ID and hypotonia in Patient 5 but not dysmorphisms, which instead resemble those of Patient 6 and the patient in study by Akilapa et [2].
For completeness, it is worth mentioning the contiguous genes deletion syndrome associated with corneal defects, mapping to the distal 12q21.33 region, and including the DCN, KERA, LUM and EPYC genes [10]. None of our patients' deletions encompassed these genes and none of them manifested corneal anomalies, in contrast to the patients described by Cano

Conclusions
We described seven novel patients with deletions scattered across the 12q21 region. 12q21 deletions are associated with a wide spectrum of clinical signs such as DD/ID, congenital malformations of the brain, heart and genitourinary systems. Considering the CR recently proposed as responsible for the most frequent clinical signs, we performed our study firstly to strengthen the role of the two major candidate genes, SYT1 and PPP1R12A, in the CR and, secondly, to investigate the effect of genes outside the CR.
Our Patients 1, 2 and 3, and others extrapolated from the DECIPHER database, carrying deletions overlapping the CR, validate the first assumption. Our Patients 5 and 6 support the hypothesis of a region outside the CR in 12q21.33, containing the ALX1 and the NTS genes, associated with dysmorphism such as wide mouth, broad nasal base and low hanging columella and ID [9].
In addition, deafness, described only for our Patient 7 and the patient from Klein et al., could be caused by haploinsufficiency of KITLG gene, since its mutation has been already identified in patients with sensorineural deafness [2,22].
Finally, since DD/ID is present in almost all patients with deletion in 12q21 regardless of the involvement of the CR, it is possible that other genes outside the CR contribute to the neurological phenotype. In particular, TMTC2, SLC6A15, CEP290, TMTC3, NTS genes, all highly expressed in the brain, could be considered as additional strong candidates [12].
In the future, investigation of novel patients with deletions in 12q21 will hopefully clarify the contribution of single genes to the complex phenotype of these patients, in order to also provide adequate counseling to patients and families in both pre-and postnatal diagnostic settings. In addition, since 12q21 microdeletions do not hamper survival and reproduction, as shown by maternal inheritance of a 5.7 Mb deletion in Patient 2, early identification of these deletions could ensure a proper intervention and supporting therapy for the patients and correct genetic counseling for the involved families and their subsequent pregnancies.
Although spreading of WES or WGS (whole genome sequencing) technologies are often crucial in identifying point mutations as the genetic cause of a disorder, sometimes proposed as first tier approaches, our work further confirms that the identification of CNVs still plays a relevant role for the diagnosis of patients with complex phenotypes and the provision of standardized psychological assessments and other medical follow-ups.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/genes13050780/s1, Table S1: List of the most relevant genes mapping to the 12q21 region.